Molecular Principles of Wnt-Frizzled Signal Initiation and Inhibition

描述（由申请人提供）：Wnt蛋白是遗传学上保守的分泌糖蛋白，其在胚胎发育和成体组织稳态期间通过经典和非经典信号传导途径调节细胞间通讯。Wnt信号传导由于其在癌症的发展和进展、组织更新和干细胞分化中的中心作用和治疗意义而被深入研究。Wnt与Frizzled（其与G蛋白偶联受体和共受体Lrp 5/6具有结构相似性）的结合对于Wnt/β-连环蛋白信号传导是不可或缺的，并且被多种内源性拮抗剂抵消。在这个提议中，我们解决了Wnt细胞外结构信息的完全缺乏，Wnt与Frizzled受体复合的方式，以及Wnt和Frizzled与内源性抑制蛋白如WIF，Dkk和Kremen的相互作用。鉴于Wnt信号传导对于多种人类疾病的核心重要性，结构信息现在变得至关重要，以便清楚地描绘受体-配体特异性的基础和受体活化的机制。该领域目前的一个困惑是Wnt、Fz和Lrp 6具有高度交叉反应性，使得将特定生物表型归因于特定Wnt-Fz受体对变得复杂。Wnt-受体复合物的结构可以通过可视化简并与配体特异性接触以及高阶信号传导复合物的结构来帮助解决这个问题。然而，获得结构数据的主要技术障碍是Wnt蛋白含有使Wnt过表达和结晶复杂化的疏水脂质修饰。在这个提议中，我们利用各种新的（酵母展示）和传统的（X射线晶体学）方法来获得参与受体激活和抑制的细胞外Wnt复合物的三维结构。我们的目标是重建几个重组Wnt-卷曲，Wnt-卷曲-Lrp 5/6和Wnt拮抗剂复合物，以表征其组装，亲和力和化学计量。我们还将尝试一个高度创新的实验，通过体外进化来生产水溶性的、非棕榈酰化的、具有生物活性的Wnt。利用X射线晶体学，我们希望阐明Wnt-卷曲-Lrp 5/6识别的结构原理，以及拮抗剂WIF，DKK和Kremen对Wnt-卷曲-Lrp 5/6复合物形成的抑制，并与Wnt领域的合作者一起，将结构数据转化为功能上有见地的实验。最终，这些雄心勃勃的研究结果将有助于揭示新的蛋白质工程策略，以询问和治疗靶向Wnt信号。 公共卫生相关性：Wnt是分泌的生长因子，其通过与卷曲G蛋白偶联受体的相互作用，与多种共受体和拮抗剂一起在胚胎发育和成体组织再生中发挥作用。Wnt信号转导代表了癌症、组织更新和干细胞分化的治疗干预的重要新前沿。我们希望获得有关Wnt-受体复合物的组装和三维结构的信息，以了解Wnt信号传导机制，并揭示新的蛋白质工程策略，以临床靶向Wnt相关疾病。

DESCRIPTION (provided by applicant): Wnt proteins are phylogenetically conserved, secreted glycoproteins that regulate cell-to-cell communication during embryonic development and adult tissue homeostasis through canonical and non-canonical signaling pathways. Wnt signaling is intensively studied due to its central role, and therapeutic implications, in the development and progression of cancer, tissue renewal, and differentiation of stem cells. Wnt binding to Frizzled, which has structural similarities to G-protein coupled receptors, and the co-receptors Lrp5/6, is indispensable for Wnt/-catenin signaling, and is counterbalanced by a variety of endogenous antagonists. In this proposal we address the complete lack of extracellular structural information on Wnts, the manner in which Wnts complex with Frizzled receptors, as well as Wnt and Frizzled interactions with endogenous inhibitory proteins such as WIF, Dkk and Kremen. Given the central importance of Wnt signaling for a variety of human diseases, structural information has now become critical in order to clearly delineate the basis of receptor- ligand specificity and mechanisms of receptor activation. A current confusion in this field is that Wnts, Fz and Lrp6 are highly cross-reactive, complicating the attribution of specific biological phenotypes to specific Wnt-Fz receptor pairs. Structures of Wnt-receptor complexes could help solve this problem by visualizing degenerate versus ligand-specific contacts, as well as the architectures of the higher order signaling complexes. However, a major technical obstacle to obtaining structural data has been that Wnt proteins contain a hydrophobic lipid modification that complicates Wnt over-expression and crystallization. In this proposal we utilize a variety of novel (yeast display) and traditional (X-ray crystallography) methodologies to obtain three-dimensional structures of extracellular Wnt complexes involved in receptor activation, and inhibition. We aim to reconstitute several recombinant Wnt-Frizzled, Wnt-Frizzled-Lrp5/6 and Wnt-antagonist complexes, in order to characterize their assemblies, affinities and stoichiometries. We will also attempt a highly innovative experiment to produce water-soluble, non-palmitoylated, bioactive Wnts by in vitro evolution. Using X-ray crystallography, we hope to elucidate the structural principles of Wnt-Frizzled-Lrp5/6 recognition, and inhibition of Wnt-Frizzled-Lrp5/6 complex formation by the antagonists WIF, DKK and Kremen, and together with established collaborators in the Wnt field, translate the structural data into functionally insightful experiments. Ultimately the results of these ambitious studies will be valuable for revealing new protein engineering strategies to interrogate and therapeutically target Wnt signaling. PUBLIC HEALTH RELEVANCE: Wnts are secreted growth factors that exert their actions in embryonic development and adult tissue regeneration through interactions with Frizzled G-protein coupled receptors, in concert with a variety of co- receptors and antagonists. Wnt signaling represents an important new frontier for therapeutic intervention in cancer, tissue renewal, and differentiation of stem cells. We wish to acquire information about the assembly and three-dimensional structures of Wnt-receptor complexes in order to understand Wnt signaling mechanisms, and reveal new protein engineering strategies to clinically target Wnt-associated diseases.